HIV-specific CTL inducing peptides and medicaments for preventing or treating AIDS comprising the peptides

ABSTRACT

Peptides which can induce specific CTLs for HIV, DNAs encoding for the peptides and medicaments for preventing and/or treating HIV comprising the peptides are provided. According to the present invention, peptides carrying novel CTL epitopes derived from HIV Pol or Env proteins are provided. These peptides can bind to HLA-A11, particularly HLA-A*1101 which is frequently encountered in Japanese people and induce HIV-specific cytotoxic T cells.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to peptides having an amino acidsequences of the partial region of the protein from HumanImmunodeficiency Virus (referred to as “HIV” hereinafter) and which caninduce an immune response against HIV, and medicaments comprising thepeptide(s) for preventing and/or treating AIDS.

BACKGROUND OF THE INVENTION

[0002] Acquired immunodeficiency syndrome (referred to as “AIDS”hereinafter) is the disease caused by the infection of HIV. Theinvestigations for medicaments to treat this disease have been activelyperformed, and although medicaments such as azidothymidine (referred toas “AZT” hereinafter) and dideoxyinosine (referred to as “DDI”hereinafter) have been practically used, they have some problems intheir effects or side effects. Thus, medicaments which can completelytreat the diseases caused by HIV infection have not been found and thereis no prospect of the findings. On the other hand, a vaccine as a toolfor preventing HIV infection and suppressing AIDS onset, which enhancesthe immune resistance against HIV, has been expected as a critical keyto control the global and rapid spread of the disease and has beenwidely investigated. To date, various types of vaccine have beeninvestigated and some of them are undergoing the clinical test.

[0003] The vaccines which have been reported to date include:

[0004] i) Vaccines which utilizes inactivated or attenuated virusparticles: The method may be considered wherein a gene responsible forHIV pathogenicity is mutated or deleted (Proc. Natl. Acad. Sci. USA, 84,1434, 1987) or the approach wherein related viruses such as simianviruses which shares the antigenecity of HIV are used (Science, 232,238, 1987), but it would be difficult to practically use them due totheir potential risks.

[0005] ii) Subunit vaccines utilizing partial antigen proteins from theviral antigenic proteins: This is the approach wherein only a partialantigenic protein of the virus particle is produce, for example, bygenetic recombinant procedures and used as an immunogen (Proc. Natl.Acad. Sci. USA, 84, 6924, (1987), Ann. Int. Med., 114,119, (1991),Nature, 355,728, 1992). This approach is widely attempted and there aremany clinical test cases. However, there are many problems such as poorincrease in the titer of the neutralizing antibody or the persistency ofthe titer of the antibody. Also, although it would be expected for thisapproach that it would be effective in enhancing the humoral immunitysuch as the production of antibodies, the effect of this approach alonewould not be necessary expected considering the infection manner of HIV,because this approach would not lead to the activation of cellularimmunity which can kill the infected cells.

[0006] iii) Recombinant live vaccines such as vaccinia virus or BCG:This is the approach wherein a partial gene sequence from HIV isintroduced into the gene of vaccinia virus (Nature, 332,728, 1988) orBCG (Nature, 351, 479, 1991) which can propagate in human cells.Theoretically, the enhancing effect on cellular immunity would beexpected but there are some problems, for example, in that at leastpreviously produced recombinant live vaccines of vaccinia did nottrigger sufficient immune response, or viruses such as vaccinia, whichare usually harmless, have the possibility of severe infection (Lancetet al. 337, 1034, 1991).

[0007] iv) Anti-idiotype antibodies: This is the approach wherein ananti-idiotype antibody is used as the alternative to the virus antigen(Proc. Natl. Acad. Sci. USA, 89, 2546, 1992).

[0008] v) Synthetic peptide vaccines: Synthesized peptides areconsidered in this approach, wherein the sequences of the determinantregion of neutralizing antibodies are chemically synthesized.

[0009] The above described vaccines are of the type of humoral immuneenhancer which mainly induce the neutralizing antibodies, while it isbelieved that cytotoxic T cells (referred to as “CTLs” hereinafter)which can impair infected cells are more important in defending theinfection than humoral immunity with neutralizing antibodies,considering that HIV spreads more easily through the cell fusion betweeninfected cells and non-infected cells than through virus particles.Actually, there is a report where if the patients who had been exposedto the chance of HIV infection were investigated, HIV specific CTLs weredetected (J Clin. Invest., 93, 1293,(1994), Nature, Med., 1, 59, 1995).There is also a report wherein HIV carrying a mutated CTL epitope canescape from the attack of CTLs (Nature Med., 3, 205, 1997) and theappearance of HIV carrying such mutated epitope is believed to be thecause of the onset of AIDS in patients chronically infected withHIV(Nature Med., 3, 212, 1997).

[0010] Antigens such as virus-derived proteins are endogenouslyprocessed in cells to short fragments and are presented on the cellsurface as a bounded form with major histocompatibility complex(referred to as “MHC” hereinafter). CTL recognizes the epitope peptidepresented by Class I MHC antigen expressed on the cell surface andattack the target cells. More specifically, CTL recognizes both of theprocessed antigen fragment bound to the groove of MHC molecule and apart of the MHC molecule on the target cell simultaneously and attackthe cell carrying them. Thus, the antigen recognition of CTL intensivelydepends on MHC on the cell surface. Such antigen recognition is calledMHC restriction. It is known that the epitope peptides which bind to aparticular MHC Class I antigen and which are presented by the antigenhave about nine amino acids in length and their amino acids sequencesare defined by certain rules (motif) (Nature, 351, 290, 1991, Eur. J.Immunol., 22, 2453, 1992, Nature, 353, 326, 1991, Nature, 360, 434,1992, Immunogenetics, 38, 161, 1993).

[0011] Based on these findings, the inventors of the present inventionhave been searching CTL peptides which specifically impair HIV-1infected cells and have already defined the peptides which can bind toHLA-B35, HLA-B51 and HLA-A31 among human Class I antigens, human Class Ileukocyte antigens (referred to as “HLA” hereinafter), and which caninduce HIV-1 specific CTLs (WO 95/11255). These peptides induced HIV-1specific CTLs, restricted by each HLA alloantigen type.

[0012] Since HLA genes are the genes having very high diversities, thepeptides which bind to HLA Class I antigens have also high diversities,although they comply with certain rules. This suggests that the peptideswhich are effective in the prevention and/or treatment of HIV infectionmay possibly differ among individuals, ethnic populations and the like.Therefore, in the prevention and/or the treatment of HIV infectionwherein such peptides are used, a set of peptides is desired which caninduce other HLA-restricted CTLs besides the known peptide groups.

SUMMARY OF THE INVENTION

[0013] It is an object of the present invention to provide a peptidewhich can induce the HIV-1 specific CTL. In particular, it is an objectof the present invention to provide a peptide which can induce HIV-1specific HLA-A*1101 restricted CTL.

[0014] It is also an object of the present invention to provide a DNAcoding for the above-described peptide. It is further the object of thepresent invention to provide a medicament for preventing and/or treatingAIDS, comprising said peptide.

[0015] It is also an object of the present invention to provide apeptide useful for developing HIV-1 vaccine, especially developing thevaccine in Asia. The major difficulty in HIV vaccine development consistin that HIV can easily mutate and escape the host immunity. Thus, thevaccine which use only one epitope as the immunogen has a greatpossibility of losing its effectiveness with time. It is an object ofthe present invention to provide multiple epitope peptides that can besimultaneously used as effective immunogens.

[0016] The peptides according to the present invention can bind toHLA-A*1101 antigen and induce HLA-A*1101 restricted CTLs targeting HIV-1infected cells. The DNAs according to the present invention encode forsaid peptides.

[0017] The inventors of the present invention have defined the peptideswhich can bind to HLA-B*3501, HLA-B*5101 and HLA-A*3101 Class I antigen(WO 95/11255), as previously mentioned. HLA is the molecule which hashigh diversities among ethnics or individuals, while the peptides of thepresent invention are characterized in that they are the peptidesderived from HIV-1 Pol or Env protein and which can bind to HLA-A11molecule frequently found in Asian people, especially in Japanese people(HLA-A*1101) (Clayton J, Lonjon C, Whittle D: Allele and haplotypefrequencies for HLA loci in various ethnic group: HLA volume I Geneticdiversity of HLA functional and medical implication: Charron D. ParisEd. : EDK Medical and Scientific International Publisher; 1997;665-820).

[0018] The peptides according to the present invention or the peptidesencoded by the DNAs according to the present invention are the fragmentsof HIV-1 Pol or Env protein, which contain any of the amino acidsequence of QIYAGIKVK (SEQ ID NO: 1), SVITQACPK (SEQ ID NO: 2),QIIEELIKK (SEQ ID NO: 3), QIIEKLIEK (SEQ ID NO: 4), ACQGVGGPSHK(SEQ IDNO: 5), AFDLSFFLK (SEQ ID NO: 6) and ALDLSHFLK (SEQ ID NO: 7) and carryHLA-A*1101 binding motif, and which can actually induce HLA-A*1101restricted CTLs against HIV-1 infected cells.

BRIEF DESCRIPTION OF DRAWINGS

[0019]FIG. 1 shows the degree of recognition of the HLA-A*1101restricted HIV-1 specific CTL epitope by the CTL clones. Blackcharacters represent C1R-A*1101 cell and white characters represent C1Rcell. The data were obtained with effector:target=2:1. Two lines of CTLclones were used for each peptide, respectively. Black circle () andwhite circle (◯) represent SF2-Pol424-9-27 CTL clone. Black triangle (▴)and white triangle (Δ) represent SF2-Pol424-9-34 CTL clone.

[0020]FIG. 2 shows the CTL activity for the target cells infected withthe recombinant vaccinia virus expressing Pol protein of HIV-1. The barswith shilling mark represents the specific lysis percentage forC1R-A*1101 infected with recombinant vaccinia virus expressing Polprotein of HIV-1. The white bar represents the specific lysis percentagefor C1R-A*1101 infected with wild type vaccinia virus.

[0021]FIG. 3 shows the CTL activity for the target cells infected withthe recombinant vaccinia virus expressing Env protein of HIV-1. The barsin FIG. 3A and 3B represent the specific cell lysis percentage forC1R-A*1101 infected with the vaccinia virus expressing Env protein,C1R-A*1101 infected with wild type vaccinia virus, C1R infected withvaccinia virus expressing Env protein, C1R infected with wild typevaccinia virus, C1R-A*1101 pulsed with SF2-Env202-9 and C1R pulsed withSF2-Env202-9, from the top to the bottom. The peptide concentration was10⁻⁶M.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The whole proteins of HIV are described, for example, in NatureVol.313, pp.277-283 (1985) or Proc. Natl., Acad. Sci. USA Vol.83,002209-2213 (1986). The peptides of the present invention are thefragments of HIV and, furthermore, they have HLA-A*1101 motif andactually bind to HLA. Exemplary HLA-A*1101 binding motif includes thepeptides consisting of 8 to 12 amino acids wherein the second amino acidthereof is selected from Val, IIe, Thr, Leu, Tyr, Cys or Phe and theC-terminal amino acid is Lys. Such peptides may be synthesized using apeptide-synthesizer. The binding of the peptides of the presentinvention containing HLA-A*1101 binding motif may be confirm by usingthe cells expressing HLA-A*1101. C1R or RMA-S cells may be used as suchcells.

[0023] According to the present invention, the synthesized peptides areconfirmed for their ability to actually stimulate the patient'speripheral blood monocytes (PBLs) and induce cytotoxic T cells (CTLs).Using such a procedure, peptides having the amino acids sequences ofQIYAGIKVK (SEQ ID NO: 1), SVITQACPK (SEQ ID NO: 2), QIIEELIKK (SEQ IDNO: 3), QIIEKLIEK (SEQ ID NO: 4), ACQGVGGPSHK (SEQ ID NO: 5). AFDLSFFLK(SEQ ID NO: 6) and ALDLSHFLK (SEQ ID NO: 7) were obtained, which are thefragments of HIV-1 Pol protein. The peptides of the present inventionmay be prepared by synthesizing them with peptide-synthesizer or byexpressing the DNAs encoding for the peptides having any of the aminoacids sequences of SEQ ID NO: 1 to SEQ ID NO: 7 with appropriatehost-vector systems. Since the codon usage may vary depending on thehost cell used, the preferred codon usage may be selected for each cell.DNAs which can hybridize with such DNAs under the stringent condition,and which bind to HLA-A11 antigen and induce cytotoxic T cells targetingHIV infected cells, may be used also. The stringent condition as usedherein means, for example, the condition wherein annealing at 20-25 ° C.below the melting temperature Tm (Tm=81.5° C.+log₁₀[Na⁺]+0.41(G+Ccontent %)−(600/sequence length)) in 2×SSC (20×SSC: NaCl 75.3 g/Lmsodium citrate 8802 g/L, pH7.0), and washing at 12-20° C. below the Tmwith salt concentration of 0.1×SSC, regarding to the temperature and thesalt concentration.

[0024] The techniques of isolation of DNA fragments, construction ofvectors, transformation using recombinant DNA technology, production ofpeptides and measurement of CTL activity are well known to those skilledin the art.

[0025] The peptide of the present invention is useful as a vaccine,because it can induce HIV-1 specific CTL functioning as the T cellepitope. As a vaccine, the peptide solution alone or the combinationthereof with one or more physiologically acceptable adjuvants may beadministrated with a syringe, or they may be administrated, for example,by aerosol, through dermal absorption from mucous membrane. The peptidemay be administrated by a single dose or multiple doses with 0.1 mg-100mg/dosage. Plural peptides may be used simultaneously, which may beeffective in certain cases. The formulation is not particularly limited,and may be freeze-dried or granulated with an excipient such as sugar.There are no acute toxicity observed for thus prepared peptideformulations according to the present invention. Adjuvants which may beused and which can increase the immunogenicity of the vaccine include acomposition from cells such as BCG cells, ISCOM extracted from QuillAdeveloped by Morein et al. (Immunostimulating complex, Nature, 308, 457,1984; Nature, 344, 873, 1990), a saponin system QS-21 (J. Immunol., 148,1438, 1992), liposome (J. Immunol., 148, 1585, 1992), alminium hidroxyde(alum), KLH (keyhole limpet hemocyanin) ‘J. Virol., 65, 489, 1991). Eachof above indicated references and Science, 255, 333, (1992) describesthat immune response such as CTL can be induced in vivo by using suchmethods.

[0026] By using the epitope peptides according to the present invention,the methods may be applicable wherein CTLs are effectively inducedwithin the body of patients by administrating the cells from thepatients or the cells carrying HLA Class I antigen of the samehaplotype, which were previously exposed to the epitope peptide, intothe vein of the patients, or wherein CTLs are induced and expanded invitro by culturing the peripheral blood lymphocytes from patients invitro with the peptide, followed by returning the cells to the patients.Therefore, CTLs obtained by culturing peripheral blood lymphocytescarrying HLA-A*1101 antigen in the presence of any of the peptideshaving the sequence according to SEQ ID NO: 1 to SEQ ID NO: 7 may beused as an AIDS vaccine.

[0027] In practice, the peptide of the present invention is added to10⁷-10⁹ peripheral blood lymphocytes of the patient and the lymphocytesare administrated into the vein of the patient after culturing for froma few hours to one day, or alternatively, after CTLs are induced bycontinuously culturing the lymphocytes in the medium supplemented with50 U/ml of recombinant IL-2 and 1 μg/ml of the peptide for a few weeks,they are injected into the vein of the patient. The cells may becultured by the methods well known to those skilled in the art, then thecultured cells are suspended, for example, in saline after the mediumcomponent is washed out, for example, by centrifugation. Suchtherapeutic methods using cell injection have been utilized as a cancertreatment, which are well known to those skilled in the art (New Ing. J.Med., 313, 4185, 1085; Science, 233, 1318,1986).

[0028] Additionally, the identified CTL epitopes according to thepresent invention may be effectively employed in vaccinia viruses or BCGlive recombinant vaccines. When the DNA encoding for the peptidescomprising any of the amino acid sequence according to SEQ ID NO: 1 toSEQ ID NO: 7 is introduced into the genes for recombinant proteins whichare intended to be expressed in the recombinant live vaccine, thepeptide will be processed in cells to be presented by HLA-A*1101 antigenafter being expressed as a part of the antigen protein. This will allowthe induction of CTL which recognize the peptide. The methods forexpressing foreign genes in recombinant BCG live vaccines are detailedin WO 88/06626. The recombinant BCG live vaccines are detailed in J.Exp. Med., 178, 197 (1993). The dose and the dosage methods may beemployed according to the dose and methods for the conventionalvariolation or BCG vaccine. They do not differ from the conventionalvariolation or BCG vaccine in the acute toxicity and the like. However,vaccinia virus should be carefully used as a therapeutic vaccine,because there may be the risk of sever infection in the patients whoseimmunocompetence was reduced by the onset of AIDS. Regarding with BCGvaccine, such cases have not been reported yet. The possibility ofinducing the immuneresponse including CTL in vivo by such methods isshown, for example, in Nature, 332, 728 (1988) and Nature, 351, 479(1991).

EXAMPLES

[0029] EXAMPLE 1

[0030] Preparation of HLA-A*1101 Expressing Cells

[0031] C1R cells expressing HLA-A*1101 (C1R-A*1101) and RMA-S cellsexpressing HLA-A*1101 (RMA-S-A*1101) were prepared as literallydescribed (Tissue Antigens, 52, 501-509,1998). RMA-S cells and C1R cellsare the mouse cell line and the human cell line, respectively, which aredefective in the transporter molecule TAP (Transporter AssociatedProtein) (Nature, 346, 476, 1990). C1R and RMA-S cells were cultured inRPMI1640 medium supplemented with 10% FCS. C1R-A*1101 and RMS-S-A*1101cells were cultured in RPM1640 medium containing 10% FCS and 0.15mg/mlHygromycin B. Epstein-Barr virus transformed cell line Tm-EBV(HLA-A11/A24, B52/B52, Cw7/Cw*1202) was maintained in RPMI1640supplemented with 10% FCS.

[0032] Unless otherwise indicated, cloning of genes, transfection ofcells and the like are performed according to the general procedures inthe filed of molecular biology (Sambrook, J. et al., Molecular Cloning:A Laboratory Manual 2nd ed. (1989), Ausbel, F. M. et al., Currentprotocols in molecular biology, John Wiley & Sons, Inc.).

EXAMPLE 2

[0033] Estimation of HLA-A*1101 Binding Peptides Derived from HIV-1-SF2Strain

[0034] 2-1. Synthesis of Peptides

[0035] Peptides were synthesized by using an automated multiple peptidessynthesizer (Shimadzu Model PSSM-8, Shimadzu Co. Kyoto Japan) and wereconfirmed by mass spectrometry. The peptides with not less than 90%purity were used in the following Examples.

[0036] 2-2. Biding Ability of the Peptides to HLA-A*1101 Molecule

[0037] Biding ability of the HIV-1-derived peptides to HLA-A*1101molecule was examined by the peptide stabilization assay as described inTissue Antigens, 52, 501-509.

[0038] The peptide stabilization assay method is described hereinafter.

[0039] RMA-S and C1R cells are the mouse cell line and the human cellline, respectively, both of which are defective in transporter moleculeTAP (Transporter Associated Protein). Therefore, they express MHC classI antigen on the cell surface at only low level when they are culturedat 37° C. However, they are known to express peptide-unbounded class Iantigen on the cell surface at high level when they are cultured at alow temperature (26° C.) (Nature, 346, 476, 1990).

[0040] Similarly, RMA-S-A*1101 and C1R-A*1101 cells express HLA-A*1101antigen on the cell surface when cultured at 26° C., but the expressionis reduced when they are cultured at 37° C. Additionally, the expressionlevel of HLA-A*1101 antigen on RMA-S-A*1101 cell or C1R-A*1101 cellpreviously cultured at 26° C. will be also reduced like the cellscultured at 37° C., by placing the cells at 37° C. for 3 hours. However,if the peptides are added externally to bind to peptide-unboundedHLA-A*1101, resulting peptide-bounded HLA-A*1101 antigen does notdisappear and remains the expression at high level even at 37° C. Thiswas used to determine the binding ability of peptide to HLA-A*1101antigen. In practice, the binding of the peptide was determined byadding a synthetic peptide to RMA-S-A*1101 or C1R-A*1101 cells culturedat 26° C., placing the cells at 26° C. for 1 hour and subsequently at37° C. for 3 hours and then determining the expression level ofHLA-A*1101 on the cell surface using flow cytometry with theanti-HLA-A*1101 monoclonal antibody. Commercially available monoclonalantibodies being capable of recognizing HLA-A antigen were used foranti-HLA-A*1101 monoclonal antibodies, but especially TP25.99 antibodywhich was a gift from Dr. Soldano Ferrone or MB40.5 antibody (ATCCHB-116) was primarily used.

[0041] As a result, 61 of 92 peptides synthesized bound to HLA-A*1101antigen molecule. HLA-A*1101 restricted CTL inducing activity wasfurther investigated for these 61 peptides.

EXAMPLE 3.

[0042] Induction of CTL from HIV-1 Infected Patients Using HLA-A*1101Binding Peptide and the Cytotoxic Activity of the CTL

[0043] CTLs were induced from HIV-1 infected patients using HLA-A*1101obtained in Example 2 and the cytotoxic activity was examined.

[0044] 3-1. Induction of CTL

[0045] Peripheral blood monocytes were prepared from LA-A*1101HIV-1carrying seropositive patients IU (HLA-A1/A24, B38/B51,Cw7/-) and KOG(HLA-A2/A11, B46/B54, C1/-). Peripheral blood monocytes were preparedaccording to the conventional Ficoll-Conray specific gravity centrifugalmethod (“New detection method of the function of lymphocytes”, JunichiYada, Michio Fujiwara ed., Chu-Gai Igaku Co., 1987; “MolecularImmunology I” of New Biochemical Experiments Course 12, Tokyo KagakuDojin, 1989). Namely, after collecting blood with heparin syringe, theblood was diluted with saline, layered on Ficoll-Paque separatingsolution (Pharmacia), followed by centrifugation at 400×g for 30minutes. Lymphocytes from intermediate layer were recovered with apipette and were used after washing. 2×10⁶ lymphocytes per well wereplaced in 24-well culture plates and cultured in RPMI640 (containing 10%FCS) supplemented with recombinant human IL-2 and a synthetic peptide atthe final concentration of 50 U/ml and 10⁻⁶M respectively. The half ofthe medium was exchanged every 2-3 day by RPMI1640 culture mediumsupplemented with 50 U/ml of recombinant human IL-2.

[0046]3-2. Determination of Cytotoxic Activity of CTL Induced by UsingHLA-A*1101 Binding Peptides (CTL assay)

[0047] Specific CTLs were induced by stimulating the bulk culture withPHA and then re-stimulating the culture by adding irradiated autologouslymphocytes (1 ×10⁶) and 10⁻⁶M of each HLA-A*1101 binding peptide every1 week. After thus re-stimulating the lymphocyte cultures for 2-3 times,the CTL activity in each culture for HLA-A*1101 expressing TM-EBVtransformed cells which had been previously pulsed with thecorresponding peptide was determined. The CTL assay was performed asdescribed thereinafter.

[0048] HLA-A*1101 expressing Tm-EBV transformed cells (1×10⁵) wereincubated for 60 minutes at 37° C. with 100 μCi of Na₂ ⁵¹CrO₄ in saline,and washed 3 times with RPMI1640 medium containing 10% FCS. 50 μl of thesuspension of labeled target cells in culture medium was added to eachwell of the 96-well plate (5×10³ cells/well). To the cells 50 μl of thesolution of each HLA-A*1101 binding peptide diluted to the concentrationof 4×10⁻⁶M-4×10⁻¹¹M was added and the cells were placed in CO₂ incubatorat 37° C. for 30 minutes. Then cultured peripheral blood cells frompatients which had been previously stimulated with the same peptide wereadded as effector cells at the effector:target (E:T) ratio of 2:1(suspended in 100 μl of medium), and the mixture was placed in a CO₂incubator at 37° C. for 4 hours.

[0049] The half of the culture medium in each of the well was taken and⁵¹Cr released from the target cells caused by the cytotoxic activity ofthe patient's cultured peripheral blood lymphocytes was determined usingγ-counter. The cytotoxic activity of CTL clone to the target cell(specific relative lysis, that is, the relative percentage of specificcell lysis) was calculated by formula (I):

Specific relative lysis (%) =(measurement of each well−minimumrelease)/(maximum release−minimum release) ×100  (i)

[0050] wherein “minimum release” is the spontaneous release of ⁵¹Cr fromtarget cells which is measured for the well containing only target cellsand “maximum release” is the release of label measured when the targetcells were disrupted by adding the surfactant Triton X-100.

[0051] As a result, the new two peptides SF2-Pol 424-9 (SEQ ID NO: 1)and SF2-Env202-9 (SEQ ID NO: 2) were obtained from at least one of thetwo patients (FIG. 1 and 3).

EXAMPLE 4

[0052] Identification of HLA-A*1101 Binding Peptides as CTL Epitopes

[0053] The two peptides SF2-Pol 424-9 (SEQ ID NO: 1) and SF2-Env202-9(SEQ ID NO:2) were confirmed to be the peptides which can actually beendogenously processed and presented by HLA-A*1101 molecule by usingisolated specific CTL clones and vaccinia virus infected cells as targetcells.

[0054] Firstly, the recombinant vaccinia virus carrying gag/pol gene orenv gene from HV-1_(SF-2) strain was generated as described in thereference (Virology, 175, 139, 1990). Cells were infected with therecombinant vaccinia virus by culturing them with 10plaque-forming-units/cell of the recombinant virus or wilt type virusover night. Obtained cells should present endogenously processed HIVgene products on the cell surface in the HLA antigen-bounded form. Onthe other hand, CTL clones specific to the two peptides SF2-Pol 424-9(SEQ ID NO: 1) and SF2-Env202-9 (SEQ ID NO: 2) were isolated frompeptide-specific bulk cultures derived from HIV infected patients,respectively.

[0055] CTL assay was performed substantially as described in Example 3-2using thus obtained vaccinia virus infected cell as target cells.Briefly, infected cells (1×10⁵) were incubated for 60 minutes with 100μCi of Na₂ ⁵¹Cr₄ in saline (at 26° C. for 90 minutes when C1R-A*1101cell or RMA-S-A*1101 cell was used), and washed three times withRPMI1640 culture medium containing 10% FCS and used as labeled targetcells. Labeled target cells (5×10³) were suspended in 50 μl of theculture medium and were added to each well of 96-well plates. To thecells 50 μl of the solution of each HLA-A*1101 binding peptide dilutedto the concentration of 4×10⁻⁶M-4×10⁻¹¹M was added and the cells wasplaced in CO₂ incubator at 37° C. for 30 minutes. HIV-Pol or Envspecific CTL clone isolated as previously described was added aseffector cells at the effector:target (E:T) ratio of 1:1-4:1 (suspendedin 100μl of medium), and the mixture was placed in a CO₂ incubator at37° C. for 4 hours. Then, the half of the culture medium in each of thewell was taken and ⁵¹Cr release from the target cells caused by thecytotoxic activity of the patient's cultured peripheral bloodlymphocytes was determined using γ-counter. Two lines of CTL wereexamined for each peptide and the cytotoxic activity of CTL clones tothe target cells was calculated by the above-described formula (I).

[0056] All the CTL clones exhibited the cytotoxic activity to C1R-A*1101which had been pulsed with the corresponding peptide (FIG. 1 and 3,effector: target=2:1). On the other hand, these CTL clones could killC1R-A*1101 cells which were infected with vaccinia virus expressingHIV-1 Pol protein or Env protein at various effector: target ratio,while they did not kill cells infected with wild type vaccinia virus(FIG. 2 and 3). These results indicate that the two peptides SF2-Pol424-9 and SF2-Env202-9 contain HLA-A*1101 restricted HIV-1 specific CTLepitopes.

EXAMPLE 5

[0057] Generality of each Epitopes in HIV-1 Infected Patients andComparison of the Ability of Inducing Specific CTL between Each Epitopes

[0058] To investigate whether the epitope SF2-Pol424-9 (SEQ ID NO: 1) ofthe present invention and two known epitopes were generally andintensively presented in HIV-1 infected patients carrying HLA-A*1101,peripheral blood monocytes (PBMCs) from six HIV-1 seropositive patientscarrying HLA-A*1101 were cultured for one week with the peptide of thepresent invention and Pol313-321 (AIFQSSMTK)(J. Immunol 159,1648-1657,197) and Pol496-505 (HIV Sequence Database, Los AlamosNational Laboratory, Los Alamos, 1997) known to be HLA-A*1101 restrictedHIV-1 specific CTL epitopes. These cells were used as effector cells,and C1R-A*1101 and C1R cells previously pulsed with the correspondingpeptide were used as target cells, to determine the CTL activity. Theresults are shown in Table 1A and 1B.

[0059] CTL activity specific for SF2-Pol424-9 and Pol496-505 was inducedin one of six patients. On the other hand, CTL activity for Pol313-321was induced in three patients. These results suggest that Pol epitopesare those among the major target molecules of HLA-A11 restricted CTL,although they may have individual differences TABLE 1A Induction of thespecific CTL by a single stimulation with the peptides in PBL fromHIV-seropositive patients carrying HLA-A11 IU TAK KOG peptide sequence40:1* 5:1 40:1 5:1 40:1 5:1 SF2-Pol424-9 QIYAGIKVK 27.4** 11.7 2.3 2.0−0.2 3.1 Pol496-505 IYQEPFKNLK 8.00 4.6 12.9 3.0 −0.6 −2.9 Pol313-321AIFQSSMTK 53.5  26.8 0.7 1.1 13.6 3.7 Env77-85*** DPNPQEVVL 2.5  1.5 0.50.0 0.2 −0.1

[0060] TABLE 1B SKG SSK SZK peptide sequence 40:1 5:1 40:1 5:1 40:1 5:1SF2- QIYAGIKVK −3.7 −1.7 7.0 −3.3 4.9 −0.2 Pol424-9 Pol496-505IYQEPFKNLK 4.9 −2.8 5.1 3.2 2.5 2.6 Pol313-321 AIFQSSMTK 2.8 −3.7 −2.42.3 31.1 8.2 Env77- DPNPQEVVL 1.7 −3.6 −0.3 −2.8 −1.1 −0.4 85***

EXAMPLE 6

[0061] The Ability of Peptides Derived from HIV-1 Subtype E and HIV-1Subtype B in Inducing Specific CTLs

[0062] Using similar procedures, epitope peptides derived from HIV-1subtype E and HIV-1 subtype B were searched to select Pol 675-9-5E(QIIEELIKK) (SEQ ID NO:3), Pol 675-9-5K8E (QIIEKLIEK) (SEQ ID NO: 4),Gag 349-11 (ACQGVGGPSHK) (SEQ ID NO: 5), Nef 84-9-2F6F (AFDLSFFLK) (SEQID NO: 6) from subtype E and Nef 84-9-2L (ALDLSHFLK) (SEQ ID NO: 7) fromsubtype B as epitope candidates. Then, human peripheral blood monocytesinfected with HIV-1 subtype E or subtype B were individually culturedwith these peptides respectively for one week and CTL activity wasdetermined by using these cells as effector cells and HLA-A*1101positive EBV-transformed cells as target cells. The results are shown inTable 3A, 3B and Table 4.

[0063] Pol 675-9-5E could induce the specific CTL from five of sevenpatients, Pol 675-9-5K8E could induce the specific CTL from two of six,Nef 84-9-2F6F could induce the specific CTL from six of seven, Gag349-11-9S could induce the specific CTL from three of seven and Nef84-9-2L could induce the specific CTL from three of five patients.Peptides from subtype E could not be recognized by corresponding subtypeB specific CTLs, and also peptides subtype E specific CTLs did notrecognize subtype B peptides. This indicated that these peptides aresubtype E specific epitopes. Additionally, among five patients who wereHLA-A11 positive and infected with HIV-1 subtype B, there was onepatient in whom the specific CTL could be induced by Nef 84-9(AVDLSHFLK) (J. Immunol 146: 1560-1565, 1991) but not by Nef 84-9-2L. Tothe contrary, there was also one patient in whom specific CTL could beinduced by Nef 84-9-2L but not by Nef 84-9 (Table 4). This makes one tobelieve that Nef 84-9-2L is the epitope distinct from Nef 84-9. TABLE 3AInduction of the specific CTL after a single stimulation with thepeptides in peripheral blood monocytes (PBLMC) from HIV-subtypeE-seropositive patients carrying HLA-A11 HIV-1 infected patient TT- TT-peptide sequence 005 TT-007 008 TT-009 Pol 675-9-5E QIIEELIKK  3.4* 55.123.4 63.8 Pol 675-9-5K8E QIIEKLIEK −4.1   21.7 10.3 4.5 Nef 84-9-2F6FAFDLSFFLK −1.3   24.6 75.5 76.5 Gag 349-11-9S ACQGVGGPSHK 4.0 84.3 82.940.4

[0064] TABLE 3B Continued from table 3A HIV-1 infected patient peptidessequence TU-002 TU-003 TU-007 Pol 675-9-5E QIIEELIKK 3.4 0.2 5.7 Pol675-9-5K8E QIIEKLIEK 5.6 −5.6 — Nef 84-9-2F6F AFDLSFFLK 15.4 41.2 64.2Gag 349-11-9S ACQGVGGPSHK 3.9 −2.8 3.4

[0065] TABLE 4 Induction of the specific CTL after a single stimulationwith the peptides in peripheral blood monocytes (PBLMC) from HIV-subtypeB-seropositive patients carrying HLA-A11 HIV-1 infected patient KI- KI-peptide sequence 005 KI-015 035 KI-030 KI-036 Nef 84-9 AVDLSHFLK 25.3*68.6 5.8 6.6 16.5 Nef 84-9-2L ALDLSHFLK 68.3  6.3 16.4 5.5 50.4

[0066] According to the present invention, novel seven epitopes of HIV-1protein and a powerful tool for development of vaccines which can induceHLA-A11 restricted CTLs are provided. The peptides according to thepresent invention can induce HIV-1 protein specific HLA-A11 restrictedCTLs. Since HLA-A11 is frequently encountered in Asian people,particularly in Japanese people, the peptides of the present inventionprovide the very important tools for the basis of AIDS investigation inAsia and for the development of HIV-1 vaccines.

1 7 1 9 PRT Human immunodeficiency virus type 1 1 Gln Ile Tyr Ala GlyIle Lys Val Lys 1 5 2 9 PRT Human immunodeficiency virus type 1 2 SerVal Ile Thr Gln Ala Cys Pro Lys 1 5 3 9 PRT Human immunodeficiency virustype 1 3 Gln Ile Ile Glu Glu Leu Ile Lys Lys 1 5 4 9 PRT Humanimmunodeficiency virus type 1 4 Gln Ile Ile Glu Lys Leu Ile Glu Lys 1 55 11 PRT Human immunodeficiency virus type 1 5 Ala Cys Gln Gly Val GlyGly Pro Ser His Lys 1 5 10 6 9 PRT Human immunodeficiency virus type 1 6Ala Phe Asp Leu Ser Phe Phe Leu Lys 1 5 7 9 PRT Human immunodeficiencyvirus type 1 7 Ala Leu Asp Leu Ser His Phe Leu Lys 1 5

What is claimed is:
 1. A peptide selected from the group of peptidesconsisting of the amino acid sequence given in SEQ ID NO: 1 to SEQ IDNO: 7 wherein said peptide can induce a cytotoxic CTL targeting to a HIVinfected cell.
 2. A DNA molecule coding for the peptide of claim
 1. 3. Amedicament for preventing and/or treating of AIDS, comprising thepeptide of claim 1, a pharmaceutically acceptable carrier and/or adiluent.